Cartilage material

ABSTRACT

Cartilage materials such as cartilage fluff and a cartilage composition comprising a particulate material are disclosed. These are suitable for stimulating chondrogenesis and/or producing cartilage regeneration. Also disclosed are processes for their preparation. Methods for regenerating articular cartilage are also disclosed, which involve, for example, placing the cartilage fluff or cartilage composition into a cartilage defect.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/006,949 filed on Jan. 14, 2011 entitled “Cartilage Material” which isa division of U.S. application Ser. No. 11/746,877 filed on May 10,2007.

BACKGROUND OF THE INVENTION

Cartilage, particularly articular cartilage, of higher animals,including man, has a limited potential for repair. Following injuries toarticular cartilage, the cartilage generally does not repair itselfbecause of its limited capacity for regeneration. The ability to repairis dependent on the extent and the depth of injury and the survivingchondrocytes and normal articular cartilage matrix. In injuriesinvolving subchondral bone, there is no regeneration of the cartilage,but there is typically enlargement and progression of the lesion withassociated pain.

The ultimate goal of treatment of these lesions is restoration of thecartilage, but in most cases this is not achieved.

Adult articular cartilage is not vascularized and, as stated above,lacks the capacity to regenerate itself after sustaining damage(Vangness, C. T., Jr. et al., Am. J. Orth. 33, No. 25S: 29, 2004). Whencartilage is cut, without involvement of subchondral bone, the defectoften will persist for the duration of the individual's life.

Treatment of articular cartilage defects may be either surgical ornon-surgical. For example, several operative procedures are currentlyused to repair or remove damaged cartilage in order to prevent furtherdestruction of the joint, decrease pain, and restore function. Theseinclude arthroscopic debridement and lavage, subchondral bonestimulating procedures, transplantation of chondrocytes or cartilageautografts and allografts and total knee arthroplasty. However, none ofthese produces regeneration of the native articular cartilage.

Thus, there is a desire for materials and methods for stimulatingchondrogenesis.

BRIEF SUMMARY OF THE INVENTION

The invention provides, in an embodiment, an animal cartilage materialin the form of a fluff. The fluff can be used for cartilageregeneration. Another embodiment of the invention provides a cartilagecomposition comprising non-demineralized particulate articular cartilagehaving large particle size, e.g., a size greater than 1,000 μm, such asabout 6,000 μm. The cartilage composition can be used for cartilageregeneration. The invention also provides processes for preparingcartilage fluff and cartilage composition.

The invention also provides a method for regenerating cartilagecomprises administering to a cartilage defect a therapeuticallyeffective amount of a cartilage fluff or a cartilage composition. Themethod may also include administering one or more additional cartilagegrowth promoting factors. In some embodiments, the inventive cartilagefluff or cartilage composition may be press-fit into a defect withoutthe use a matrix or without the use of a cover to contain the material.

The present invention provides one or more significant features. Forexample, the present invention provides a cartilage fluff or a cartilagecomposition with demonstrable chondrogenic activity. The cartilage fluffor cartilage composition according to the invention induces newcartilage formation in articular cartilage defects. The cartilage fluffadvantageously packs the articular defect without the use of a matrix orvehicle, e.g., glycerol, collagen gel, etc., in combination with thefluff. The cartilage fluff and cartilage composition of the inventionshow complete or substantially complete regeneration of articularcartilage defects. When placed in a cartilage defect, the inventivecartilage fluff and cartilage composition produce new cartilage growthcongruitous with the articular surface, e.g., in defects involvingsubchondral bone. Furthermore, new cartilage formation may be initiatedrapidly. In embodiments, new cartilage formation begins within, a shortperiod of time, e.g., two weeks, and complete, or substantially completecoverage of the defect may occur by about sixteen weeks.

Advantageously, embodiments of the present invention avoid the difficultprocess steps of extracting growth factors and proteoglycans and/orsimilar substances from cartilage with various chemical agents.Embodiments of the present invention produce effective cartilage fluffand cartilage compositions without deleteriously altering the growthfactors and other substances present in the cartilage.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with an embodiment, the present invention provides acartilage fluff. The cartilage fluff can be used for stimulatingchondrogenesis and/or producing cartilage regeneration. The cartilagefluff comprises articular cartilage material in the form of a cartilagefluff.

The present invention in an embodiment provides a cartilage fluffcomprising an animal, e.g., human, cartilage material in the form of afluff. The cartilage fluff comprises strands of animal cartilagematerial with the strands having an average length of about 200 μm ormore, e.g., about 400 μm or more, about 600 μm or more, about 800 μm ormore, about 1,000 μm or more, about 1,200 μm or more, about 1,400 μm ormore, about 1,600 μm or more, about 1,800 μm or more, about 2,000 μm ormore, about 2,200 μm or more, about 2,400 μm or more, about 2,600 μm ormore, about 2,800 μm or more, to about 3,000 μm. Alternatively, or inaddition, the average length can be about 2,900 μm or less, about 2,700μm or less, about 2,500 pm or less, about 2,300 pm or less, about 2,100μm or less, about 1,900 μm or less, about 1,700 pm or less, about 1,500μm or less, about 1,300 pm or less, about 1,100 μm or less, about 900 μmor less, about 700 μm or less, about 500 μm or less, to about 300 pm orless. Preferably, the cartilage fluff has strands having an averagelength of about 1,000 μm to about 3,000 μm. More preferably, thecartilage fluff has strands having an average length from about 2,000 μmto about 3,000 μm.

Additionally, in embodiments of the invention, the cartilage fluffcomprises strands of animal, e.g., human, cartilage material with thestrands having an average width of about 30 μm or more, e.g., about 40μm or more, about 50 μm or more, about 60 μm or more, about 80 pm ormore, about 100 μm or more, about 120 pm or more. Alternatively, or inaddition, the average width can be about 140 μm or less, about 130 μm orless, about 110 μm or less, about 90 μm or less, about 70 μm or less,about 50 μm or less, about 40 μm or less. Preferably, the cartilagefluff comprises strands of animal cartilage material with strands havingan average width of about 40 μm to about 140 μm.

The cartilage fluff of the invention has one or more branches that areintertwined with one or more of the strands. The cartilage fluffcomprises strands of animal, e.g., human, cartilage material with thestrands having one or more branches of an average diameter up to about50 μm. Preferably, the cartilage fluff comprises strands having one ormore branches of an average diameter from about 5 μm or more, e.g.,about 10 μm, about 20 μm, about 30 μm, about 40 μm, to about 50 μm.Alternatively, or in addition, the average diameter can be about 45 μmor less, about 35 μm or less, about 25 μm or less, about 15 μm or less,to about 5 μm. Preferably, the cartilage fluff comprises strands havingone or more branches of an average diameter from about 5 μm to about 50μm, more preferably, from about 10 μm to about 30 μm.

A significant property of the cartilage fluff is that it allows for thematerial to be packed efficiently into articular and bone defectswithout the need to mix it with a vehicle such as glycerol, collagengel, etc. In an embodiment, the strands of cartilage in the cartilagefluff are intertwined, and thus form a matrix which supports itself. Thematrix supports itself and will thus remain in the articular defect intowhich the cartilage fluff is placed. The physical configuration of thecartilage strands allow for release of cartilage regeneration factorfrom the strands, thus promoting efficient or rapid cartilageregeneration. Cartilage fluff encourages retention of clotted bloodbetween the interwoven strands and promotes cartilage regeneration.Physically, in this respect, cartilage fluff resembles a cottoncomposition.

In another embodiment, the invention provides a cartilage compositioncomprising non-demineralized particulate cartilage having a particlesize greater than 1000 μm. The particulate cartilage may have an averageparticle size from 1,100 pm or more, e.g., about 1,500 μm or more, about2,000 μm or more, about 2,500 μm or more, about 3,000 pm or more, about3,500 pm or more, about 4,000 μm or more, about 4,500 μm or more, about5,000 pm or more, about 5,500 μm or more, to about 6,000 μm.Alternatively, or in addition, the average particle size can be about5,800 μm or less, about 5,300 μm or less, about 4,800 μm or less, about4,300 μm or less, about 3,800 μm or less, about 3,300 μm or less, about2,800 μm or less, about 2,300 pm or less, about 1,800 μm or less, toabout 1,300 pm or less. Preferably, the particulate cartilage has anaverage particle size from an average size from about 3,000 pm to about6,000 μm, more preferably an average particle size from about 4,000 μmto about 6,000 μm, and even more preferably an average particle sizefrom about 5,000 μm to about 6,000 μm.

Articular cartilage material may be obtained from any suitable source,e.g., articular surfaces of joints, such as from distal femurs, proximaltibias, acetabulums, heads of femurs, and/or heads of radiuses. Thecartilage may be removed, for example, with a scalpel blade andpreferably removed down to subchondral bone, without removing bone. Thearticular cartilage for use in the present invention may includearticular hyaline cartilage, fibrocartilage, and mixtures thereof. Thearticular cartilage for use in the present invention may compriseallogeneic cartilage, xenogeneic cartilage, and mixtures thereof.Allogeneic cartilage may be obtained from a human cadaver. Xenogeneiccartilage may be obtained from a variety of animals. The articularcartilage for use in the present invention may comprise human cartilage.Preferably, the cartilage fluff allograft may be obtained from a humancadaver. Preferably, the cartilage fluff xenograft may be may beobtained from a variety of animals.

The articular cartilage is preferably non-demineralized. Preferably, thecartilage is not subjected to harsh chemical treatments, which can alterthe inherent natural properties of cartilage material. For example, thecartilage is preferably not subjected to demineralization treatmentssuch as treatment with hydrochloric acid, ethylene diamine, and/or otherdemineralization agents. The non-demineralized articular cartilage mayoptionally be subjected to microbiological testing or subjected to othertesting protocols that do not deleteriously alter the cartilage.

Additionally, the articular cartilage is preferably not subjected to anyphysical treatment that may demineralize and/or alter the inherentnatural properties of the cartilage. For example, the articularcartilage is preferably not subjected to elevated temperatures, e.g.,temperatures greater than about 50° C., that may diminish thechondrogenic activity of the cartilage. The articular cartilage may bepreserved, e.g., freeze-dried, frozen, cryo-preserved, and/or dried,after being removed from the joint. One preferred method of preservingarticular cartilage is freeze-drying.

The cartilage material used in embodiments of the invention may be inthe form of dry cartilage, freeze-dried cartilage, frozen cartilage,cryo-preserved cartilage, wet cartilage, or mixtures thereof. Thecartilage for use in the present invention may include the samecartilage materials as previously disclosed in paragraph [0017]. In anembodiment, the cartilage is freeze-dried. For example, pieces ofcartilage obtained from the articular surface of one or more joints iswashed in normal saline, preferably several changes of normal saline,blotted dry, and frozen rapidly, e.g., at 10° C./min or faster, in thevapor phase of liquid nitrogen (about −150° C.) or alternatively in theliquid phase of liquid nitrogen (about −196° C.). After being frozen,the cartilage is preferably rapidly placed directly on the shelves of afreeze-drying apparatus maintained at about −40° C. to about −50° C.(the condenser being cooled from about −70° C. to about −80° C.). Avacuum level of less than about 100 millitorr is preferably maintainedin the freeze-drying chamber during the freeze-drying cycle. Thefreeze-drying cycle may last an average of about 5 days. During theinitial 30-45 minutes of the cycle, the cartilage warms from the initialfrozen temperature (e.g., about −150° C.) to the temperature of thefreeze-drying chamber (e.g., about −40° C.), after which it ismaintained at about −40° C. for the remainder of the cycle. Preferably,the moisture content of the cartilage is reduced to about 5% or less,preferably to about 4% or less, e.g., to about 3% to about 5%. Overdrying is preferably avoided, as this may result in the irreversiblealterations of collagen and proteoglycan structures. At the end of thefreeze-drying cycle, the chamber is warmed to room temperature, thevacuum released and the freeze-dried cartilage is removed.

According to the invention, the articular cartilage may be ground ormilled using any suitable grinding or milling apparatus. For example,any grinding or milling apparatus capable of grinding dry, hard, brittlematerial in seconds, such as turbo mills, disc mills, toothed discmills, or jet mills are suitable for grinding or milling the cartilage.

Preferably, the grinding and milling are performed under conditions thatpreclude raising the temperature of the articular cartilage to a levelthat may diminish the chondrogenic activity of the cartilage material.For example, grinding is preferably performed without raising thetemperature of the articular cartilage above about 50° C. Inembodiments, grinding is preferably performed without raising thetemperature of the cartilage above about 40° C. The temperature of thearticular cartilage may be measured in any suitable manner. For example,thermocouples may be used to monitor the temperature of the cartilagedirectly, e.g., by measuring the temperature of the cartilageimmediately after grinding, or indirectly, e.g., by measuring thetemperature of the metal in the grinding mill. For example, continuousgrinding in conventional grinding mills for 3-5 minutes can raise thetemperature of the material to 70° C. or above. Continuous grinding ormilling may be coupled with adequate cooling to keep the temperature ata desired level, e.g., at a temperature of 2° to 4° C. Operating agrinding mill intermittently may preclude an undesirable rise intemperature. In one embodiment, freeze-dried pieces of cartilage may beground in a grinding mill operating intermittently for less than oneminute, e.g., for about 20 to about 30 seconds, with intervals betweengrinding. Such intervals can be from about 30 to about 120 seconds.

Another method of preparing a cartilage fluff according to the presentinvention comprises freeze-drying cartilage to obtain a freeze-driedcartilage, grinding the freeze-dried cartilage to obtain a groundmaterial comprising cartilage fluff, and separating the cartilage flufffrom the ground material.

Preferably, after each grinding cycle, the cartilage may be sieved. Thecartilage may be sieved through sieves of 850 μm and 1,000 μm size.Material remaining on the sieve exceeds the pore size of the sieve.Accordingly, the cartilage material may have an average length aspreviously disclosed in paragraph [0012]. The cartilage fluff inaccordance with an embodiment of the invention has a cohesivecharacteristic in that the fluff sticks to each other to form a network,e.g., a continuous network. This unique property allows for easy packingand treatment of an articular defect.

Another method of preparing a cartilage fluff according to the inventioncomprises freeze-drying articular cartilage, and milling thefreeze-dried cartilage. For example, the milling may be carried out witha drill bit, core drill, or burr. The cartilage fluff according to theinvention may be prepared by securing bone with cartilage in a millingand digitizing machine, and shaving the cartilage to obtain thecartilage fluff. Alternatively, the articular cartilage may beunattached to bone.

For example, the milling of the cartilage can be performed incrementallyin an industrial type mill without over heating the cartilage.Overheating is prevented by employing milling cycles of 15-30 seconds.Alternatively, the chamber of the mill is cooled, e.g., by carbondioxide, cryogenic gases or by placing the entire milling apparatus intoa refrigerated chamber. Periodically, the milled material is placed intosieves and smaller particles are segregated by passing through thesieves with openings of 500 μm or less.

In accordance with another embodiment of the invention, the cartilagefluff may be prepared from freeze-dried cartilage by the use of adigitizing and milling machine, such as a Levil type. A freeze-driedcartilage block with or without bone is placed on the milling platformand the machine is activated. Preferably, after each milling cycle,small particles are sieved out of the cartilage being milled. Thecartilage being milled may be placed into cryogenic gas vapor or othercold chamber to maintain it in a frozen state. The frozen cartilage maybe replaced into the milling chamber and subjected to another cycle ofmilling. This process can be repeated several times as needed.

A method of preparing a particulate cartilage composition comprisesfreeze-drying cartilage to obtain a freeze-dried cartilage, grinding thefreeze-dried cartilage to obtain a ground material comprising aparticulate cartilage composition, and separating the particulatecartilage composition from the ground material. After each grindingcycle, the cartilage may be sieved. The cartilage may be sieved throughsieves of 25 μm to 6,000 μm. Sieving may be used to separate cartilageinto particle sizes of less than 1,100 μm and cartilage particle sizesof 1,100 μm to about 6,000 μm. Grinding may be repeated until anydesired or preferred cartilage particle sizes are obtained.

The present invention further provides a method of regeneratingarticular cartilage in an animal, e.g., human, in need thereofcomprising administering an effective amount of the cartilage fluff ofthe invention to a site of the animal where there is a need forregenerating articular cartilage. Therapeutically effective amounts ofcartilage composition comprising an articular cartilage fluff of theinvention may be administered at the site of a cartilage defect. Thecartilage fluff may be implanted at the site, e.g., articular surface,and packed into the defect. In embodiments, the cartilage fluff may bepacked without the use of a vehicle, which is advantageous for rapidregeneration of articular cartilage.

The present invention also provides a method of regenerating articularcartilage in an animal, e.g., human, in need thereof comprisingadministering an effective amount of a cartilage composition of theinvention to a site of the animal where there is a need for regeneratingarticular cartilage. Therapeutically effective amounts of a cartilagecomposition comprising particulate cartilage having an average particlesize of from 1,100 μm to about 6,000 μm may be administered at the siteof a cartilage defect. The particulate cartilage composition may beimplanted at the site, e.g., articular surface and packed into thedefect. In embodiments, the cartilage composition may be packed into thedefect with the use of an overlying cover.

The method may also include administering one or more additionalcartilage growth promoting factors. Additives may be applied to thecartilage fluff or cartilage compositions in order to increasechondrocyte migration and proliferation. The cartilage fluff andcartilage compositions can support the addition of a variety ofchondrogenic stimulating factors including, but not limited to growthfactors (FGF-2, FGF-5, IGF-1, TGF-.beta., BMP-2, BMP-7, PDGF, VEGF),human allogenic or autologous chondrocytes, human allogenic orautologous bone marrow cells, stem cells, demineralized bone matrix,insulin, insulin-like growth factor-1, transforming growth factor-B,interleukin-1 receptor antagonist, hepatocyte growth factor,platelet-derived growth factor, Indian hedgehog and parathyroidhormone-related peptide or bioactive glue.

Without wishing to be bound to any theory, it is believed that cartilagefluff and cartilage compositions of the invention release cartilagegrowth factor(s) or other favorable substances that induce or enhanceregeneration of articular cartilage. The three-dimensional shape of thefluff and the particles and multiple surfaces, as well as the inventivefluff sizes and particle sizes, enhance diffusion of the cartilagegrowth factor(s) or other substances from the fluff and the particles.Furthermore, the absence of harsh chemical treatments and avoidance ofelevated temperatures during processing facilitates the production offluff and particles having high chondrogenic activity.

The following example further illustrates the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLE

This example demonstrates the chondrogenic activity of the cartilagefluff in fully immunocompetent non-human primates. Full thicknesscartilage defects measuring 10 mm-×−1 mm is created in the medialcondyles of the animals. The defects are densely packed with thecartilage fluff according to the present invention and are compactedwith a tamp. The animals are examined two, six, and sixteen weeks posttransplantation and the joints are re-explored. Photographs taken attwo, six, and sixteen weeks, respectively. Specimens are also taken andfixed in 10% formalin-Earle's balanced salt solutions. Paraffin sectionsare cut and stained with homotoxylin and eosin, PAS, Romanowski-Giemsaand Safranin-O stains.

At two weeks post transplantation, granulation tissue is present in thecenter of the defect and new cartilage is present at the edge of thedefect. At six weeks post transplantation, new cartilage is formed fromthe edges of the defect and granulation tissue is no longer present andthe cartilage composition is covered with a translucent membrane in thecenter of the defect. At six weeks, the hyaline cartilage shows normalmorphology. At sixteen weeks post transplantation, the defect iscompletely healed and covered with congruous normal appearing cartilage.At sixteen weeks, normal appearing hyaline cartilage is formed. Thecontrol, where the defect is left untreated, shows no evidence ofhealing; the defect becomes larger due to additional fragmentation ofthe cartilage.

All references, including publications, patent applications, andpatents, cited hen are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein are merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range, unless otherwise indicated herein, andeach separate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventor for carrying out the invention. Ofcourse, variations of those preferred embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventor intends for the invention tobe practiced otherwise than as specifically described herein.Accordingly, this invention includes all modification equivalents of thesubject matter recited in the claims appended hereto as permitted byapplicable law. Moreover, any combination of the-above-describedelements in all possible variations thereof is encompassed by theinvention unless otherwise indicated herein or otherwise clearlycontradicted by context.

What is claimed is:
 1. A cartilage composition for repairing articularcartilage defects comprises: an articular cartilage material, thematerial being non-demineralized and not subjected to demineralizationtreatments or harsh chemicals which alter the natural properties of thecartilage material, the cartilage material in the form of pieces ofcartilage having been washed in normal saline washes and frozen rapidly,not having been subjected to elevated temperatures greater than 50degrees C. is dried to about 3 to 5 percent moisture content, the piecesof cartilage material being preserved, freeze-dried, frozen orcryo-preserved cartilage; the pieces of preserved cartilage material isground or milled or shaved intermittently without raising thetemperature above 40 degrees C. to form a ground, milled or shavedmaterial of a cartilage fluff and cartilage particles, after eachgrinding or milling cycle, the material is sieved through sieve openingsof 850 μm and 1000 μm size allowing the cartilage particles to pass anda cartilage fluff made of a plurality of branched strands to beseparated from the particles, the cartilage fluff having cohesivecharacteristics wherein the fluff sticks to itself to form a continuousnetwork of cartilage fluff for packing into defects of articularcartilage or bone.
 2. The cartilage composition for repairing articularcartilage defects of claim 1 wherein the strands have one or morebranches, the strands having an average length up to 3000 μm.
 3. Thecartilage composition for repairing articular cartilage defects of claim2 wherein the strands have an average width of 40 μm to 140 μm.
 4. Thecartilage composition for repairing articular cartilage defects of claim3 wherein the one or more branches of the strands have an averagediameter of 5 μm to 50 μm.
 5. The cartilage composition for repairingarticular cartilage defects of claim 1 wherein the strands of cartilagein the cartilage fluff are intertwined.
 6. The cartilage composition forrepairing articular cartilage defects of claim 5 wherein the intertwinedstrands form a self-supporting matrix having a physical configurationresembling a cotton composition.
 7. The cartilage composition forrepairing articular cartilage defects of claim 6 wherein the cartilagefluff has the physical configuration of the cartilage strands allowingfor a release of cartilage regeneration factors from the strands topromote rapid cartilage regeneration in articular cartilage defects. 8.The cartilage composition for repairing articular cartilage defects ofclaim 1 wherein the articular cartilage material is an allogeniccartilage from a human cadaver.
 9. The cartilage composition forrepairing articular cartilage defects of claim 1 wherein the articularcartilage is a human cartilage.
 10. The cartilage composition forrepairing articular cartilage defects of claim 9 wherein the humancartilage is obtained from a human cadaver.
 11. The cartilagecomposition for repairing articular cartilage defects of claim 1 whereinthe articular cartilage material is a xenogenic cartilage obtained froman animal.
 12. The cartilage composition for repairing articularcartilage defects of claim 1 wherein the allograft cartilage material isone or more of allogenic cartilage, xenogenic cartilage, allograftcartilage or mixtures thereof.
 13. The cartilage composition forrepairing articular cartilage defects of claim 1 wherein the articularcartilage material is obtained from articular surfaces of joints. 14.The cartilage composition for repairing articular cartilage defects ofclaim 1 wherein the articular cartilage includes articular hyalinecartilage, fibrocartilage and mixtures thereof.
 15. The cartilagecomposition for repairing articular cartilage defects of claim 1 whereinthe articular cartilage fluff is packed into a defect without using avehicle, such as glycerol, collagen gel, the matrix formed by thearticular cartilage fluff supports itself and remains in the articulardefect into which it is placed.
 16. A method of preparing a cartilagecomposition comprises the steps of: a) obtaining an articular cartilagematerial, the material being in the form of pieces of cartilagenon-demineralized and not subjected to demineralization treatments orharsh chemicals which alter the material properties of the cartilagematerial and not subjecting the material to elevated temperaturesgreater than 50 degrees C.; b) washing the cartilage material in normalsaline using several washes; c) rapidly freezing and drying the materialto about 3 to 5 percent moisture content; d) preserving the pieces ofcartilage material by freeze drying, freezing or cryo-preserving; e)intermittently grinding, milling or shaving the preserved pieces ofarticular cartilage material without raising the material temperatureabout 40 degrees C. to form a ground, milled or shaved material of acartilage fluff and cartilage particles; f) sieving the cartilage fluffand particles through a sieve after each intermittent grinding; g)allowing the cartilage particles to pass; and h) collecting thecartilage fluff made of a plurality of branched strands to be formed inthe sieve, the cartilage fluff having a cohesive characteristic whereinthe cartilage fluff sticks to itself to form a continuous network ofcartilage fluff for packing into defects in articular cartilage or bone.17. The method of claim 16 further comprises the step of: administeringan effective amount of the cartilage fluff to a site of the animal orhuman where there is a need for regenerating articular cartilage. 18.The method of claim 12, wherein the cartilage fluff is applied withoutthe use of a vehicle.
 19. The method of claim 16 further comprises thestep of: comprising freeze drying cartilage to obtain a freeze-driedcartilage, grinding the freeze-dried cartilage to obtain a groundmaterial comprising cartilage fluff and cartilage particles, andseparating the cartilage fluff from the cartilage particles of theground material.
 20. The method of claim 16 further comprises the stepof: securing bone with cartilage in a milling and digitizing machine,and shaving the cartilage to obtain the cartilage fluff.
 21. The methodof claim 20, wherein the shaving is carried out with a drill bit, coredrill, or burr.
 22. The method of claim 16 further comprises the stepof: freeze-drying articular cartilage and milling the freeze-driedcartilage to obtain the cartilage fluff.
 23. The method of claim 22,wherein said articular cartilage is attached to bone.
 24. The method ofclaim 22, wherein said articular cartilage is unattached to bone. 25.The method of claim 16 further comprises the step of: processing flufffrom frozen cartilage or from cryopreserved cartilage.
 26. The method ofclaim 17 further comprises the step of: administering one or moreadditional cartilage growth promoting factors.
 27. The method of claim26 further comprises the step of: adding one or more of chondrogenicstimulating factors to the cartilage fluff including, growth factorsFGF-2, FGF-5, IGF-1, TGF-.beta., BMP-2, BMP-7, PDGF, VEGF, humanallogenic or autologous chondrocytes, human allogenic or autologous bonemarrow cells, stem cells, demineralized bone matrix, insulin,insulin-like growth factor-1, transforming growth factor-B,interleukin-1 receptor antagonist, hepatocyte growth factor,platelet-derived growth factor, Indian hedgehog and parathyroidhormone-related peptide or bioactive glue.
 28. The method of claim 16,wherein the step of sieving includes using a sieve having 850 μm to 1000μm openings.
 29. The method of claim 28, wherein the sieved articulatedfluff is in the range of 1000 μm to 3000 μm.